The prognostic value of neutrophil-to-lymphocyte ratio in cholangiocarcinoma: a systematic review and meta-analysis

The neutrophil-to-lymphocyte ratio (NLR) is used as biomarker in malignant diseases showing significant association with poor oncological outcomes. The main research question of the present study was whether NLR has also prognostic value in cholangiocarcinoma patients (CCA). A systematic review was carried out to identify studies related to NLR and clinical outcomes in CCA evaluating the literature from 01/2000 to 09/2021. A random-effects model, pooled hazard ratios (HR) and 95% confidence interval (CI) were used to investigate the statistical association between NLR and overall survival (OS) as well as disease-free survival (DFS). Subgroup analyses, evaluation of sensitivity and risk of bias were further carried out. 32 studies comprising 8572 patients were eligible for this systematic review and meta-analysis. The pooled outcomes revealed that high NLR prior to treatment is prognostic for poor OS (HR 1.28, 95% CI 1.18–1.38, p < 0.01) and DFS (HR 1.39, 95% CI 1.17–1.66, p < 0.01) with meaningful HR values. Subgroup analysis revealed that this association is not significantly affected by the treatment modality (surgical vs. non-surgical), NLR cut-off values, age and sample size of the included studies. Given the likelihood of NLR to be prognostic for reduced OS and DFS, pre-treatment NLR might serve as a useful biomarker for poor prognosis in patients with CCA and therefore facilitate clinical management.


Material and methods
Literature search. The ex-ante protocol of this systematic review was registered open access in the International Prospective Register of Systematic Reviews (PROSPERO) under the ID: CRD42021271435 and was conducted in line with recommendations of the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-analyses) statement. PubMed and Google Scholar were systematically searched for articles published between January 2000 and September 2021. The following key search terms were used: "lymphocytes" OR "Neutrophil-to-lymphocyte ratio" AND "Cholangiocarcinoma (CCA)" OR "Biliary tree cancers (BTC)". Two independent literature searches were carried out by two authors based on the same strategy. Subsequently, no further publications were identified after the reference list and citations search were completed.
Inclusion and exclusion criteria. Inclusion criteria were: • Studies investigating the prognostic value of NLR in CCA.
Exclusion criteria were: • No access to the full text. • Reviews, case reports, comments or editorials.
Statistical analysis. The statistical analysis was conducted as previously described 7 . Hazard ratios (HR) and 95% CIs were used to assess the association between NLR and outcomes. Kaplan-Meier curves in combination with Engauge Digitizer version 12.1 were used to extract these information if not directly reported as described previously 14 . RevMan version 5.4 and R project version 4.1.2 were used to analyze and visualize the results. Measures of statistical heterogeneity between studies were calculated (tau, Q, I value) and assessed using the Chi-squared test and assumed to be significant when I 2 > 50% and/or p < 0.05. A random-effect model and subgroup analysis were preferred when heterogeneity existed, while a fixed-effect model was used when no variance was detected in the data set. Subgroup analysis was carried out to investigate heterogeneity in the studies, while sensitivity analyses were performed to determine the stability of the overall effects. Here, one study at a time was excluded to ensure that no single study would dominate and would be solely responsible for a significant result. Baujat plots were used to investigate the contribution of studies to the heterogeneity as well as pooled outcome and funnel plots were utilized to evaluate publication bias 15 . Quality assessment of selected studies. The quality of the included studies was structurally evaluated by 2 reviewers (DL and JB) using the Newcastle-Ottawa scale 16 . The Newcastle-Ottawa scale is composed of the following three quality indicators: outcome assessment, comparability, and selection. Each paper was scored from 0 to 9 based on these parameters.

Results
Literature search. The process of selecting publications is depicted in Fig. 1 Study characteristics and quality assessment. The key characteristics of the 32 publications analyzed in this manuscript are depicted in Table 1. All publications included were retrospective cohort studies comprising a total of 8572 patients, 6427 of whom had liver resection and 2145 of whom had undergone non-surgical therapy. The mean age of the study populations was 56 to 70 years with males accounting for 31% to 79% of the patients in the investigated data set. NLR cut-off values were different between the studies and obtained using various approaches. Regarding the investigated entities, 19 studies focused on ICCA, 8 on ECCA and 5 studies analyzed both ICCA and ECCA. While all studies reported a correlation between OS and NLR, only 14 reported a correlation between DFS and NLR. The study quality was evaluated between six and nine points on the Newcastle-Ottawa quality assessment scale indicating that the methodology of the investigations was of generally good quality (Table 2).

Sensitivity analyses of correlation between NLR and prognosis of CCA patients.
To determine the prognostic robustness of NLR, a random effects model in sensitivity analyses was adopted, deleting each study in each turn. As shown in Fig. 3, the results of the pooled HRs changed in each analysis, but high NLR still displayed an unfavorable effect on OS and DFS. These results indicate that the association between NLR and survival in CCA is certainly robust.
Contribution of studies to the heterogeneity and pooled outcome. Baujat Fig. S3B).

Publication bias.
No bias influencing the HRs could be detected as the results from a funnel plot analysis displayed no asymmetry ( Supplementary Fig. S4).

Discussion
Recently, a number of studies have investigated the interaction between inflammation and cancer 8,49 . NLR, as an inflammatory index, has been shown to be associated with various clinical endpoints including long-term prognosis, disease recurrence and response to treatment 50 . Previous meta-analyses demonstrated that high NLR Table 2. Qualities of cohort studies are evaluated by modified Newcastle-Ottawa scale. The quality of the included studies was assessed under six items of Hayden et al. All included translational studies reporting oncological outcome were evaluated in accordance with the Newcastle-Ottawa scale. The maximum score of the scale is nine points with studies being categorized as low (0-3 points), moderate (4-6 points) and high quality (7-9 points), respectively.
Chronic inflammation is believed to play a notable role in 15% of cancer cases globally and it is accepted that a systemic inflammatory activation is an important player in carcinogenesis and progression 60 . However, the underlying mechanisms explaining how NLR influences oncological outcomes are yet to be explored 7 . In the clinical setting, systemic inflammation is primarily reflected and quantified by changes in blood parameters and can be determined by the counts of various cell components of the peripheral blood (neutrophils, lymphocytes, monocytes and platelets) using standard thresholds 61 . Previous studies have already shown that the association between neutrophils and circulating tumor cells (CTCs) drives cell cycle progression to confer proliferative advantage of CTC clusters, leading to faster metastasis development and enhanced metastatic potential of CTC clusters 62 .
Local and systemic inflammation is often involved in the initial carcinogenesis, cell proliferation, angiogenesis, and metastasis or progression of malignant tumors 6 . Quail et al. have linked neutrophilia to tumor-derived granulocyte colony-stimulating factor (GCSF) which at the same time accelerates tumor development 63 . Other studies depicted that neutrophils itself promote the survival and proliferation of malignant cells by secreting pro-inflammation mediators, such as tumor necrosis factor alpha (TNFa), interleukin (IL) 1, IL 6 and vascular endothelial growth factor 64 . Furthermore, a meta-analysis on NLR in solid tumors in general also demonstrated that high NLR is associated with poor survival in many malignancies, showing a particularly pronounced effect in metastatic advanced disease 8 . Park et al. also found that an elevated NLR is associated with a poor lymphocytemediated cytotoxicity against tumor cells characterized by a lower density of tumor-infiltrating lymphocytes (CD3+ and CD8+ T cells) in individuals with colorectal cancer 65 .
Escape from immune surveillance is considered to be a key characteristic of tumorigenesis and cancer progression. Novel treatment modalities, e.g. immune checkpoint inhibitors (ICIs), tumor vaccines and chimeric antigen receptor (CAR) T-cells are currently under investigation and suggested to have high potential to improve treatment 66 . However, in contrast to other solid tumors, the response rates to immunotherapy have not shown satisfying results which may be attributed to the spatial heterogeneity in CCA per se. In fact, there is a lack of reliable prognostic biomarkers and risk-assessment tools which would be suitable to predict the future response to these therapies. This is also considered a main obstacle in the use of immunotherapies in CCA patients. Katayama et al. studied NLR in 81 patients diagnosed with non-small cell lung cancer (NSCLC) who received atezolizumab as monotherapy and observed that patients with high NLR at baseline exhibited shorter progression-free survival and OS compared to those with a low NLR 67 . Li et al. reported that patients receiving ICIs for metastatic disease with NLR < 5 showed significantly longer OS 68 . In addition, Ota et al. studied the data of 98 patients who received nivolumab and found that poor prognostic factors of OS were pretreatment NLR of > 3 and NLR difference of > 2 over 60 days before and after receiving nivolumab. Those individuals with NLR difference > 2 displayed a longer median OS 69 . Hence, NLR holds promise to predict treatment response to ICIs in CCA as well.
The pure prognostic value of NLR was frequently investigated in other tumor entities. Yang et al. conducted a meta-analysis based 1804 pancreatic cancer patients and showed that high NLR was linked to reduced OS in individuals treated by chemotherapy or surgical resection. Furthermore, a high NLR was associated to tumor metastasis, poor tumor differentiation, poor performance status and elevated carbohydrate antigen 19-9 56 . Moreover, NLR indicated reduced OS and DFS in breast cancer patients, with its prognostic value being retained across different clinicopathologic parameters such disease stage and subtypes 70 . In patients with esophageal cancer, a higher pretreatment NLR was linked to shorter survival as well as deeper tumor invasion and the presence of lymph node metastases 53 . Surprisingly, ethnicity had also an impact on certain studies. For example, Gu et al. discovered that NLR has consistent prognostic value in metastatic castration-resistant prostate cancer patients and predicts poor PFS/RFS in Asian, but not in Caucasian individuals 71 . In colon cancer patients undergoing a variety of treatments such as resection of the primary tumor, palliative chemotherapy and resection or ablation for liver metastases, higher preoperative NLR was prognostic for a lower survival rate 72 . Given these reports in other solid tumors, our results were in line with previous results and support a general role NLR as prognostic in solid malignancies.
As all meta-analyses with limited available studies, the analysis has certainly limitations due to the lack of high-level evidence: www.nature.com/scientificreports/ • The study comprised a variety of methodologies and, most importantly, different NLR cutoff levels.
• Further, the included studies were retrospective in nature and therefore have an inherent potential of selection bias. As several studies did not explicitly report HRs and CIs, these variables were extrapolated from the Kaplan-Meier curves in some papers 27,28,36,38,46,48 . • A detailed investigation of the association between NLR and tumor clinicopathological characteristics was not feasible as the published data were unfortunately not detailed enough. This also accounts for the different types of cholangiocarcinoma as some studies include both ICCA and ECCA in a unified analysis as well as the distinct molecular subtypes of CCA.
Future research should therefore focus on the role of NLR in different subtypes and the identification of a uniform NLR cut-off to facilitate the implantation into clinical management of patients. Despite these obvious limitations, the present meta-analysis has also inherent strengths: • Representative data set especially for patients undergoing surgical treatment.
• Detailed sub-analysis for study sample size, age, NLR cut-off value, geographical region, tumor subtypes and different types. • The inclusion of a sensitivity analysis indicating that no single study is responsible for the overall significant effect of NLR on OS and DFS.

Conclusions
Considering the aforementioned limitations and the limited available sample size, this study indicates a notable likelihood of NLR to be prognostic for reduced OS and DFS. Elevated NLR before treatment might therefore serve as biomarker for reduced oncological outcome (OS and DFS) in CCA patients. As patients undergoing surgery for CCA display high rates of perioperative morbidity and mortality, preoperative patient selection is fundamental to balance surgical risk with oncological benefits. Here, NLR provides additional information for treatment selection and risk stratification.